Lyssavirus antigen constructs
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example 2
Expression of SAM Lyssaviral Antigens
[0342]Western blot analysis was performed to determine whether the transgenes were expressed from the SAM rabies constructs. BHK cells (1×106) were transfected with 2.5 ug RNA from Constructs 1-4. After 20 hours, cell extracts were harvested and the production of rabies G protein was analysed by SDS gel electrophoresis followed by western blot analysis with the mouse anti-rabies glycoprotein antibody MAB8727 (Millipore Sigma, Billerica Mass., US). After incubation with primary antibody, the membrane was washed and then incubated with the peroxidase-conjugated anti-mouse antibody 115-035-003 (Jackson ImmunoResearch Laboratories, Inc., West Grove Pa., US). Finally the assay was developed by electrochemiluminescence (ECL) using standard techniques (GE Healthcare RPN2106, Little Chalfont, UK).
[0343]FIG. 2A demonstrates that BHK cells transfected with Constructs 1-4 all expressed rabies G protein. Molecular weight standards showing the location of 39 ...
example 3
mmunogenicity
[0345]The immunogenicity of the SAM rabies constructs was evaluated in parallel with RABAVERT in BALB / c mice (FIG. 3A and FIG. 3B). Each of the four constructs shown in FIG. 1A and Table 1 was formulated with either a cationic nanoemulsion (CNE) or a lipid nanoparticle (LNP). The animals were immunized on days 0 and 21 with the SAM vaccine constructs and on days 0, 7 and 21 with RABAVERT, according to the licensed RABAVERT dosing schedule. Each group consisted of 10 mice. Sera were collected on days 14 and 35 to evaluate the animals' immunogenicity against rabies. Serology was performed by the micro rapid Fluorescent Focus Inhibition Test for rabies (RFFIT) to determine the titer of anti-rabies neutralizing antibodies (nAb) (Smith et al. Bull. World Health Organ. 48:535 (1973)). The World Health Organization (WHO) guidelines recommend using the RFFIT to determine nAb titers and considers an Ab titer of 0.5 IU / ml to be an adequate response to a rabies vaccine (WHO Positi...
example 4
s Vaccines Provide Long-Term Immunogenicity
[0349]The ability of SAM rabies vaccines to confer long term immunogenicity was examined and the results are shown in FIG. 4. Construct 4 was formulated in amounts of 1.5 ug with CNE (square), 0.15 ug with LNP (triangle) or 1.5 ug with LNP (inverted triangle). RABAVERT (circle) was administered in three doses on days 0, 7 and 21 at a dilution factor of 1 / 25 of the human clinical dose. Neutralizing antibody titers of mice immunized with each of these formulations were measured by RFFIT at days 56, 90 and 180 days post-immunization. The dotted line denotes the threshold of immunogenicity for a rabies vaccine of 0.5 IU / ml.
[0350]At day 14, 1.5 ug Construct 4 RNA formulated with CNE, 0.15 ug Construct 4 RNA formulated with LNP or 1.5 ug Construct 4 RNA formulated with LNP elicited neutralizing antibodies to rabies at levels well above the immunogenicity threshold of effectiveness. By day 35 and at subsequent time points, the LNP-formulated SAM v...
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